Engineered cell entry links receptor biology with single-cell genomics.

Cells communicate with each other via receptor-ligand interactions. Here, we describe lentiviral-mediated cell entry by engineered receptor-ligand interaction (ENTER) to display ligand proteins, deliver payloads, and record receptor specificity. We optimize ENTER to decode interactions between T cell receptor (TCR)-MHC peptides, antibody-antigen, and other receptor-ligand pairs. A viral presentation strategy allows ENTER to capture interactions between B cell receptor and any antigen. We engineer ENTER to deliver genetic payloads to antigen-specific T or B cells to selectively modulate cellular behavior in mixed populations. Single-cell readout of ENTER by RNA sequencing (ENTER-seq) enables multiplexed enumeration of antigen specificities, TCR clonality, cell type, and states of individual T cells. ENTER-seq of CMV-seropositive patient blood samples reveals the viral epitopes that drive effector memory T cell differentiation and inter-clonal vs. intra-clonal phenotypic diversity targeting the same epitope. ENTER technology enables systematic discovery of receptor specificity, linkage to cell fates, and antigen-specific cargo delivery.

Evolving topological order in the postnatal visceral pleura.

BACKGROUND: Changes in epithelial cell shape reflects optimal cell packing and the minimization of surface free energy, but also cell-cell interactions, cell proliferation, and cytoskeletal rearrangements. RESULTS: Here, we studied the structure of the rat pleura in the first 15 days after birth. After pleural isolation and image segmentation, the analysis demonstrated a progression of epithelial order from postnatal day 1 (P1) to P15. The cells with the largest surface area and greatest shape variability were observed at P1. In contrast, the cells with the smallest surface area and most shape consistency were observed at P15. A comparison of polygonal cell geometries demonstrated progressive optimization with an increase in the number of hexagons (six-sided) as well as five-sided and seven-sided polygons. Analysis of the epithelial organization with Voronoi tessellations and graphlet motif frequencies demonstrated a developmental path strikingly distinct from mathematical and natural reference paths. Graph Theory analysis of cell connectivity demonstrated a progressive decrease in network heterogeneity and clustering coefficient from P1 to P15. CONCLUSIONS: We conclude that the rat pleura undergoes a striking change in pleural structure from P1 to P15. Further, a geometric and network-based approach can provide a quantitative characterization of these developmental changes.

United States-Based Colorectal Cancer Surgical Trials Lack Representation and Adequate Reporting of Racially and Ethnically Diverse Participants: Systematic Review and Regression Analysis.

BACKGROUND: Despite the established National Institute of Health Revitalization Act, which aims to include ethnic and racial minority representation in surgical trials, racial and ethnic enrollment disparities persist. OBJECTIVE: To assess the proportion of patients from minority races and ethnicities that are included in colorectal cancer surgical trials and reporting characteristics. DATA SOURCES: Search was performed using MEDLINE (Ovid), Embase, Web of Science, and Cochrane Central. STUDY SELECTION: Inclusion criteria included 1) trials performed in the United States between January 1, 2000, and May 30, 2022; 2) patients with colorectal cancer diagnosis; and 3) surgical intervention, technique, or postoperative outcome. Trials evaluating chemotherapy, radiotherapy, or other nonsurgical interventions were excluded. INTERVENTIONS: Pooled proportion and regression analysis was performed to identify the proportion of patients by race and ethnicity included in surgical trials and the association of year of publication and funding source. MAIN OUTCOME MEASURES: Proportion of trials reporting race and ethnicity and proportion of participants by race and ethnicity included in surgical trials. RESULTS: We screened 10,673 unique publications, of which 80 were examined in full text. Fifteen studies met our inclusion criteria. Ten (66.7%) trials did not report race, 3 reported races as a proportion of White participants only, and 3 reported 3 or more races. There was no description of ethnicity in 11 (73.3%) trials, with 2 describing "non-Caucasian" as ethnicity and 2 describing only Hispanic ethnicity. Pooled proportion of White participants was 81.3%, of Black participants was 6.2%, of Asian participants was 3.6%, and of Hispanic participants was 3.5%. LIMITATIONS: A small number of studies was identified that reported racial or ethnic characteristics of their participants. CONCLUSIONS: Both race and ethnicity are severely underreported in colorectal cancer surgical trials. To improve outcomes and ensure the inclusion of vulnerable populations in innovative technologies and novel treatments, reporting must be closely monitored.

Topography of pleural epithelial structure enabled by en face isolation and machine learning.

Pleural epithelial adaptations to mechanical stress are relevant to both normal lung function and parenchymal lung diseases. Assessing regional differences in mechanical stress, however, has been complicated by the nonlinear stress-strain properties of the lung and the large displacements with ventilation. Moreover, there is no reliable method of isolating pleural epithelium for structural studies. To define the topographic variation in pleural structure, we developed a method of en face harvest of murine pleural epithelium. Silver-stain was used to highlight cell borders and facilitate imaging with light microscopy. Machine learning and watershed segmentation were used to define the cell area and cell perimeter of the isolated pleural epithelial cells. In the deflated lung at residual volume, the pleural epithelial cells were significantly larger in the apex (624 ± 247 µm2 ) than in basilar regions of the lung (471 ± 119 µm2 ) (p < 0.001). The distortion of apical epithelial cells was consistent with a vertical gradient of pleural pressures. To assess epithelial changes with inflation, the pleura was studied at total lung capacity. The average epithelial cell area increased 57% and the average perimeter increased 27% between residual volume and total lung capacity. The increase in lung volume was less than half the percent change predicted by uniform or isotropic expansion of the lung. We conclude that the structured analysis of pleural epithelial cells complements studies of pulmonary microstructure and provides useful insights into the regional distribution of mechanical stresses in the lung.

Cell-Penetrating Peptides for Use in Development of Transgenic Plants.

Genetically modified plants and crops can contribute to remarkable increase in global food supply, with improved yield and resistance to plant diseases or insect pests. The development of biotechnology introducing exogenous nucleic acids in transgenic plants is important for plant health management. Different genetic engineering methods for DNA delivery, such as biolistic methods, Agrobacterium tumefaciens-mediated transformation, and other physicochemical methods have been developed to improve translocation across the plasma membrane and cell wall in plants. Recently, the peptide-based gene delivery system, mediated by cell-penetrating peptides (CPPs), has been regarded as a promising non-viral tool for efficient and stable gene transfection into both animal and plant cells. CPPs are short peptides with diverse sequences and functionalities, capable of agitating plasma membrane and entering cells. Here, we highlight recent research and ideas on diverse types of CPPs, which have been applied in DNA delivery in plants. Various basic, amphipathic, cyclic, and branched CPPs were designed, and modifications of functional groups were performed to enhance DNA interaction and stabilization in transgenesis. CPPs were able to carry cargoes in either a covalent or noncovalent manner and to internalize CPP/cargo complexes into cells by either direct membrane translocation or endocytosis. Importantly, subcellular targets of CPP-mediated nucleic acid delivery were reviewed. CPPs offer transfection strategies and influence transgene expression at subcellular localizations, such as in plastids, mitochondria, and the nucleus. In summary, the technology of CPP-mediated gene delivery provides a potent and useful tool to genetically modified plants and crops of the future.

Matrix metalloproteinase activity and prostaglandin E2 are elevated in the synovial fluid of meniscus tear patients.

PURPOSE: Meniscus tears are a common knee injury and are associated with the development of post-traumatic osteoarthritis (OA). The purpose of this study is to evaluate potential OA mediators in the synovial fluid and serum of meniscus tear subjects compared to those in the synovial fluid of radiographic non-OA control knees. MATERIALS AND METHODS: Sixteen subjects with an isolated unilateral meniscus injury and six subjects who served as reference controls (knee Kellgren-Lawrence grade 0-1) were recruited. Twenty-one biomarkers were measured in serum from meniscus tear subjects and in synovial fluid from both groups. Meniscus tear subjects were further stratified by tear type to assess differences in biomarker levels. RESULTS: Synovial fluid total matrix metalloproteinase (MMP) activity and prostaglandin E2 (PGE2) were increased 25-fold and 290-fold, respectively, in meniscus tear subjects as compared to reference controls (p < 0.05). Synovial fluid MMP activity and PGE2 concentrations were positively correlated in meniscus tear subjects (R = 0.83, p < 0.0001). In meniscus tear subjects, synovial fluid levels of MMP activity, MMP-2, MMP-3, sGAG, COMP, IL-6, and PGE2 were higher than serum levels (p < 0.05). Subjects with complex meniscus tears had higher synovial fluid MMP-10 (p < 0.05) and reduced serum TNFα and IL-8 (p < 0.05) compared to other tear types. CONCLUSIONS: Given the degradative and pro-inflammatory roles of MMP activity and PGE2, these molecules may alter the biochemical environment of the joint. Our findings suggest that modulation of PGE2 signaling, MMP activity, or both following a meniscus injury may be targets to promote meniscus repair and prevent OA development.

Chronic Exertional Compartment Syndrome in Athletes.

Chronic exertional compartment syndrome (CECS) refers to exercise-induced, reversible increases in pressure within well-defined inelastic fascial compartments leading to compromised tissue perfusion followed by functional loss, ischemic pain, and neurologic symptoms. Symptoms typically resolve when the activity ceases and there are usually no permanent sequelae. In the upper extremity, this condition most commonly affects athletes during sports requiring repetitive and vigorous gripping, such as rowers. In addition to clinical history and examination, a number of methods aid diagnosis, including compartment pressure measurements, magnetic resonance imaging, and near infrared spectroscopy. When symptoms persist despite conservative treatment, multiple operative techniques have been described to treat CECS including open, mini-open, and endoscopic release of involved compartments. We review the pathophysiology, diagnostic modalities, treatment strategies, and outcomes data for CECS of the upper extremity while highlighting areas of residual controversy.

Loss of stiffness in collagen-rich uterine fibroids after digestion with purified collagenase Clostridium histolyticum.

BACKGROUND: Uterine fibroids are a significant health problem. These common benign tumors occur in 70-80% of women before age 50 years and often cause bleeding and pain and can interfere considerably with daily life. Current treatment options are limited. Fibroids contain substantial amounts of altered and disordered collagens, which contribute to their bulk. Targeting these collagens directly presents a novel treatment approach. OBJECTIVES: We sought to test the hypothesis that a highly purified collagenase Clostridium histolyticum will digest interstitial collagen in uterine fibroids and reduce their stiffness and thereby evaluate the feasibility that this collagenase C histolyticum can be developed into an alternative treatment for fibroids. A secondary objective was to describe the collagen content of the fibroid tissue. STUDY DESIGN: Fibroid tissue cubes (1 cm3; n = 154) were cut from 17 uterine fibroids that were obtained from 7 consented subjects undergoing scheduled hysterectomies. Tissue cubes were injected with diluent, placebo, or highly purified collagenase C histolyticum (0.05, 0.1, or 0.2 mg/cube) and incubated at 37°C for 24, 48, 72, or 96 hours. At each time point, 6 noninjected control cubes were also evaluated. Tissue cubes were photographed before and after incubation. Myometrial samples (n = 21) were also evaluated. Stiffness was quantified through rheometry by measuring complex shear moduli of the tissues. Percent fibrosis was determined by computerized analysis of Masson-trichrome-stained slides. Digestion of collagen fibrils was confirmed by transmission electron microscopy. RESULTS: Fibrosis in untreated fibroids ranged from 37% to 77%, reflecting the collagen-rich nature of these tumors. After treatment with collagenase for 96 hours, fibrosis ranged from 5.3% to 2.4%. Transmission electron microscopy confirmed complete digestion of collagen fibrils. Tissue stiffness was reduced with all 3 doses of collagenase treatment and at all 4 time points. Longer incubation times with collagenase caused greater reduction in stiffness, and treated cubes lost their cuboidal shape and had gelatinous/liquefied centers. At 96 hours the stiffness in tissues treated with the lowest dose was reduced to 966 ± 106 Pascal compared with the diluent-treated control at the same time (5323 ± 903 Pascal; P < .0001; by analysis of variance with Tukey-Kramer). CONCLUSION: Uterine fibroids have a high content of collagen that can be effectively digested by highly purified collagenase C histolyticum, resulting in reduced tissue stiffness. Loss of stiffness may decrease bulk symptoms in vivo and possibly lead to shrinkage of fibroids through changed mechanotransduction, leading ultimately to reduced fibroid symptoms of pain and bleeding. Clinical trials are necessary to evaluate the safety and efficacy of collagenase C histolyticum including the rate of regrowth of fibroids. The data of this study provide a strong rationale for using this purified collagenase in clinical trials as a local treatment for women with fibroids.

Comparative mechanisms of protein transduction mediated by cell-penetrating peptides in prokaryotes.

Bacterial and archaeal cell envelopes are complex multilayered barriers that serve to protect these microorganisms from their extremely harsh and often hostile environments. Import of exogenous proteins and nanoparticles into cells is important for biotechnological applications in prokaryotes. In this report, we demonstrate that cell-penetrating peptides (CPPs), both bacteria-expressed nona-arginine peptide (R9) and synthetic R9 (SR9), are able to deliver noncovalently associated proteins or quantum dots into four representative species of prokaryotes: cyanobacteria (Synechocystis sp. PCC 6803), bacteria (Escherichia coli DH5α and Arthrobacter ilicis D-50), and archaea (Thermus aquaticus). Although energy-dependent endocytosis is generally accepted as a hallmark that distinguishes eukaryotes from prokaryotes, cellular uptake of uncomplexed green fluorescent protein (GFP) by cyanobacteria was mediated by classical endocytosis. Mechanistic studies revealed that macropinocytosis plays a critical and major role in CPP-mediated protein transduction in all four prokaryotes. Membrane damage was not observed when cyanobacterial cells were treated with R9/GFP complexes, nor was cytotoxicity detected when bacteria or archaea were treated with SR9/QD complexes in the presence of macropinocytic inhibitors. These results indicate that the uptake of protein is not due to a compromise of membrane integrity in cyanobacteria, and that CPP can be an effective and safe carrier for membrane trafficking in prokaryotic cells. Our investigation provides important new insights into the transport of exogenous proteins and nanoparticles across the complex membrane systems of prokaryotes.

Three Arginine-Rich Cell-Penetrating Peptides Facilitate Cellular Internalization of Red-Emitting Quantum Dots.

Nanoparticles, such as semiconductor quantum dots (QDs), have been found increasing use in biomedical diagnosis and therapeutics because of their unique properties, including quantum confinement, surface plasmon resonance, and superparamagnetism. Cell-penetrating peptides (CPPs) represent an efficient mechanism to overcome plasma membrane barriers and deliver biologically active molecules into cells. In this study, we demonstrate that three arginine-rich CPPs (SR9, HR9, and PR9) can noncovalently complex with red light emitting QDs, dramatically increasing their deliv- ery into living cells. Zeta-potential and size analyses highlight the importance of electrostatic interactions between positive-charged CPP/QD complexes and negative-charged plasma membranes indicating the efficiency of transmembrane complex transport. Subcellular colocalization indicates associations of QD with early endosomes and lysosomes following PR9-mediated delivery. Our study demonstrates that nontoxic CPPs of varied composition provide an effective vehicle for the design of optimized drug delivery systems.

Pseudopapilledema and association with idiopathic intracranial hypertension.

PURPOSE: Diagnosing idiopathic intracranial hypertension (IIH), or pseudotumor cerebri, can be challenging in children. Diagnosis is based on lumbar puncture, opening pressures, and appearance of the optic disk. Misdiagnosis of papilledema, a typical finding, may lead to unnecessary treatments and procedures. We report 52 children over a 6-year period to better identify the true incidence of pseudopapilledema and other factors that may confound the diagnosis of IIH. METHODS: A retrospective chart review approved by the Institutional Review Board was performed. Fifty-two children under the age of 21 referred to us based on suspected IIH or papilledema from 2007 to 2013 are included in this study. Patients were assessed by a pediatric ophthalmologist and a neurosurgeon. RESULTS: Fifty-two children were initially diagnosed with IIH and/or papilledema; 26 diagnoses were revised to pseudopapilledema after pediatric ophthalmological review. Out of those 26 patients with pseudopapilledema, 14 had undergone lumbar punctures, 19 had MRIs, 9 had CTs, and 12 were taking medications-these medications were discontinued upon revision of the diagnoses. The difference in the CSF opening pressure between children diagnosed with true IIH (32.7 cm H2O) and children diagnosed with pseudopapilledema (24.7 cm H2O) was statistically significant. CONCLUSIONS: IIH diagnosis is heavily reliant on the appearance of the optic disk. Pediatric ophthalmological assessment is essential to carefully examine the optic disk and prevent further unnecessary investigation and treatments. Close communication between pediatricians, ophthalmologists, and neurosurgeons can avoid invasive procedures for children who do have pseudopapilledema, and not IIH or associated papilledema.

Group peer mentoring to improve faculty connections and enhance mentoring networks.

BACKGROUND: Difficulty finding mentors and forging connections in academic departments can be challenging and became even more so when the COVID-19 pandemic reduced opportunities for informal networking. Even as restrictions on in-person meetings eased, many faculty preferred meetings to remain virtual. Because some of the most powerful predictors of faculty vitality are positive professional relationships and feelings of inclusion and belonging to an institution, attending to faculty needs in this area is important to mitigate undesired lingering consequences. APPROACH: We created structured peer mentoring groups for our department's physicians and psychologists that meet virtually. Groups span career stages, academic appointments and clinical interests. The purpose was to establish a deeper culture of mentoring, increase feelings of connection to a supportive community within the department, facilitate career planning and enhance the development of skills necessary in academic medicine such as teaching skills, scholarly productivity and personal wellness. EVALUATION: A survey conducted after the first year of the programme was completed by 70% of eligible faculty (31/45). Ninety-six percent felt the programme had created an inclusive and appreciative culture, 86% met faculty members they had never met before and 79% sought mentoring advice from a colleague they would not usually have interacted with in that manner. All participants appreciated hearing their colleagues' perspectives on topics they do not typically discuss. IMPLICATIONS: Departmentally based group peer mentoring that spans career stages and interests can facilitate faculty connections and enhance a supportive culture of mentorship.

Buckling forces and the wavy folds between pleural epithelial cells.

Cell shapes in tissues are affected by the biophysical interaction between cells. Tissue forces can influence specific cell features such as cell geometry and cell surface area. Here, we examined the 2-dimensional shape, size, and perimeter of pleural epithelial cells at various lung volumes. We demonstrated a 1.53-fold increase in 2-dimensional cell surface area and a 1.43-fold increase in cell perimeter at total lung capacity compared to residual lung volume. Consistent with previous results, close inspection of the pleura demonstrated wavy folds between pleural epithelial cells at all lung volumes. To investigate a potential explanation for the wavy folds, we developed a physical simulacrum suggested by D'Arcy Thompson in On Growth and Form. The simulacrum suggested that the wavy folds were the result of redundant cell membranes unable to contract. To test this hypothesis, we developed a numerical simulation to evaluate the impact of an increase in 2-dimensional cell surface area and cell perimeter on the shape of the cell-cell interface. Our simulation demonstrated that an increase in cell perimeter, rather than an increase in 2-dimensional cell surface area, had the most direct impact on the presence of wavy folds. We conclude that wavy folds between pleural epithelial cells reflects buckling forces arising from the excess cell perimeter necessary to accommodate visceral organ expansion.

Mechanistic studies of intracellular delivery of proteins by cell-penetrating peptides in cyanobacteria.

BACKGROUND: The plasma membrane plays an essential role in selective permeability, compartmentalization, osmotic balance, and cellular uptake. The characteristics and functions of cyanobacterial membranes have been extensively investigated in recent years. Cell-penetrating peptides (CPPs) are special nanocarriers that can overcome the plasma membrane barrier and enter cells directly, either alone or with associated cargoes. However, the cellular entry mechanisms of CPPs in cyanobacteria have not been studied. RESULTS: In the present study, we determine CPP-mediated transduction efficiency and internalization mechanisms in cyanobacteria using a combination of biological and biophysical methods. We demonstrate that both Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 strains of cyanobacteria possess red autofluorescence. Green fluorescent protein (GFP), either alone or noncovalently associated with a CPP comprised of nine arginine residues (R9/GFP complexes), entered cyanobacteria. The ATP-depleting inhibitor of classical endocytosis, N-ethylmaleimide (NEM), could block the spontaneous internalization of GFP, but not the transduction of R9/GFP complexes. Three specific inhibitors of macropinocytosis, cytochalasin D (CytD), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and wortmannin, reduced the efficiency of R9/GFP complex transduction, indicating that entry of R9/GFP complexes involves macropinocytosis. Both the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) and membrane leakage analyses confirmed that R9/GFP complexes were not toxic to the cyanobacteria, nor were the endocytic and macropinocytic inhibitors used in these studies. CONCLUSIONS: In summary, we have demonstrated that cyanobacteria use classical endocytosis and macropinocytosis to internalize exogenous GFP and CPP/GFP proteins, respectively. Moreover, the CPP-mediated delivery system is not toxic to cyanobacteria, and can be used to investigate biological processes at the cellular level in this species. These results suggest that both endocytic and macropinocytic pathways can be used for efficient internalization of regular protein and CPP-mediated protein delivery in cyanobacteria, respectively.

Geometric and network organization of visceral organ epithelium.

Mammalian epithelia form a continuous sheet of cells that line the surface of visceral organs. To analyze the epithelial organization of the heart, lung, liver and bowel, epithelial cells were labeled in situ, isolated as a single layer and imaged as large epithelial digitally combine montages. The stitched epithelial images were analyzed for geometric and network organization. Geometric analysis demonstrated a similar polygon distribution in all organs with the greatest variability in the heart epithelia. Notably, the normal liver and inflated lung demonstrated the largest average cell surface area (p < 0.01). In lung epithelia, characteristic wavy or interdigitated cell boundaries were observed. The prevalence of interdigitations increased with lung inflation. To complement the geometric analyses, the epithelia were converted into a network of cell-to-cell contacts. Using the open-source software EpiGraph, subgraph (graphlet) frequencies were used to characterize epithelial organization and compare to mathematical (Epi-Hexagon), random (Epi-Random) and natural (Epi-Voronoi5) patterns. As expected, the patterns of the lung epithelia were independent of lung volume. In contrast, liver epithelia demonstrated a pattern distinct from lung, heart and bowel epithelia (p < 0.05). We conclude that geometric and network analyses can be useful tools in characterizing fundamental differences in mammalian tissue topology and epithelial organization.

Biomechanics of a Plant-Derived Sealant for Corneal Injuries.

Purpose: The corneal epithelium has a glycocalyx composed of membrane-associated glycoproteins, mucins, and galactin-3. Similar to the glycocalyx in visceral tissues, the corneal glycocalyx functions to limit fluid loss and minimize frictional forces. Recently, the plant-derived heteropolysaccharide pectin has been shown to physically entangle with the visceral organ glycocalyx. The ability of pectin to entangle with the corneal epithelium is unknown. Methods: To explore the potential role of pectin as a corneal bioadhesive, we assessed the adhesive characteristics of pectin films in a bovine globe model. Results: Pectin film was flexible, translucent, and low profile (80 µm thick). Molded in tape form, pectin films were significantly more adherent to the bovine cornea than control biopolymers of nanocellulose fibers, sodium hyaluronate, and carboxymethyl cellulose (P < 0.05). Adhesion strength was near maximal within seconds of contact. Compatible with wound closure under tension, the relative adhesion strength was greatest at a peel angle less than 45 degrees. The corneal incisions sealed with pectin film were resistant to anterior chamber pressure fluctuations ranging from negative 51.3 ± 8.9 mm Hg to positive 214 ± 68.6 mm Hg. Consistent with these findings, scanning electron microscopy demonstrated a low-profile film densely adherent to the bovine cornea. Finally, the adhesion of the pectin films facilitated the en face harvest of the corneal epithelium without physical dissection or enzymatic digestion. Conclusions: We conclude that pectin films strongly adhere to the corneal glycocalyx. Translational Relevance: The plant-derived pectin biopolymer provides potential utility for corneal wound healing as well as targeted drug delivery.

Pectin based biologic Velcro effectively seals traumatic solid organ and small bowel injuries.

INTRODUCTION: Injuries to the liver and small bowel are common in multiple injuries. While there are currently a variety of accepted damage-control techniques to expeditiously manage such injuries, morbidity and mortality remain high. Pectin polymers have previously been shown to effectively seal visceral organ injuries ex vivo through physiochemical entanglement with the glycocalyx. We sought to compare the standard of care for the management of penetrating liver and small bowel injuries with a pectin-based bioadhesive patch in a live animal model. METHODS: Fifteen adult male swine underwent a laparotomy with standardized laceration to the liver. Animals were randomized to one of three treatment arms: packing with laparotomy pads (n = 5), suture repair (n = 5), or pectin patch repair (n = 5). Following 2 hours of observation, fluid was evacuated from the abdominal cavity and weighed. Next, a full-thickness small bowel injury was created, and animals were randomized to either a sutured repair (n = 7) or pectin patch repair (n = 8). The segment of bowel was then pressurized with saline, and the burst pressure was recorded. RESULTS: All animals survived the protocol to completion. There were no clinically significant differences between groups regarding baseline vitals or laboratory studies. On one-way analysis of variance, there was a statistically significant difference between groups regarding blood loss after liver repair (26 mL suture vs. 33 mL pectin vs. 142 mL packing, p < 0.01). On post hoc analysis, there was no statistically significant difference between suture and pectin ( p = 0.9). After repair, small bowel burst pressures were similar between pectin and suture repair (234 vs. 224 mm Hg, p = 0.7). CONCLUSION: Pectin-based bioadhesive patches performed similarly to the standard of care for the management of liver lacerations and full-thickness bowel injuries. Further testing is warranted to assess the biodurability of a pectin patch repair, as it may offer a simple option to effectively temporize traumatic intra-abdominal injuries.

Bioadhesive patch as a parenchymal sparing treatment of acute traumatic pulmonary air leaks.

INTRODUCTION: Traumatic pulmonary injuries are common in chest trauma. Persistent air leaks occur in up to 46% of patients depending on injury severity. Prolonged leaks are associated with increased morbidity and cost. Prior work from our first-generation pectin patches successfully sealed pulmonary leaks in a cadaveric swine model. We now test the next-generation pectin patch against wedge resection in the management of air leaks in anesthetized swine. METHODS: A continuous air leak of 10% to 20% percent was created to the anterior surface of the lung in intubated and sedated swine. Animals were treated with a two-ply pectin patch or stapled wedge resection (SW). Tidal volumes (TVs) were recorded preinjury and postinjury. Following repair, TVs were recorded, a chest tube was placed, and animals were observed for presence air leak at closure and for an additional 90 minutes while on positive pressure ventilation. Mann-Whitney U test and Fisher's exact test used to compare continuous and categorical data between groups. RESULTS: Thirty-one animals underwent either SW (15) or pectin patch repair (PPR, 16). Baseline characteristics were similar between animals excepting baseline TV (SW, 10.3 mL/kg vs. PPR, 10.9 mL/kg; p = 0.03). There was no difference between groups for severity of injury based on percent of TV loss (SW, 15% vs. PPR, 14%; p = 0.5). There was no difference in TV between groups following repair (SW, 10.2 mL/kg vs. PPR, 10.2 mL/kg; p = 1) or at the end of observation (SW, 9.8 mL/kg vs. PPR, 10.2 mL/kg; p = 0.4). One-chamber intermittent air leaks were observed in three of the PPR animals, versus one in the SW group ( p = 0.6). CONCLUSION: Pectin patches effectively sealed the lung following injury and were noninferior when compared with wedge resection for the management of acute traumatic air leaks. Pectin patches may offer a parenchymal sparing option for managing such injuries, although studies evaluating biodurability are needed.

3D stimulated Raman spectral imaging of water dynamics associated with pectin-glycocalyceal entanglement.

Pectin is a heteropolysaccharide responsible for the structural integrity of the cell walls of terrestrial plants. When applied to the surface of mammalian visceral organs, pectin films form a strong physical bond with the surface glycocalyx. A potential mechanism of pectin adhesion to the glycocalyx is the water-dependent entanglement of pectin polysaccharide chains with the glycocalyx. A better understanding of such fundamental mechanisms regarding the water transport dynamics in pectin hydrogels is of importance for medical applications, e.g., surgical wound sealing. We report on the water transport dynamics in hydrating glass-phase pectin films with particular emphasis on the water content at the pectin-glycocalyceal interface. We used label-free 3D stimulated Raman scattering (SRS) spectral imaging to provide insights into the pectin-tissue adhesive interface without the confounding effects of sample fixation, dehydration, shrinkage, or staining.

A proteome-scale map of the SARS-CoV-2-human contactome.

Understanding the mechanisms of coronavirus disease 2019 (COVID-19) disease severity to efficiently design therapies for emerging virus variants remains an urgent challenge of the ongoing pandemic. Infection and immune reactions are mediated by direct contacts between viral molecules and the host proteome, and the vast majority of these virus-host contacts (the 'contactome') have not been identified. Here, we present a systematic contactome map of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with the human host encompassing more than 200 binary virus-host and intraviral protein-protein interactions. We find that host proteins genetically associated with comorbidities of severe illness and long COVID are enriched in SARS-CoV-2 targeted network communities. Evaluating contactome-derived hypotheses, we demonstrate that viral NSP14 activates nuclear factor κB (NF-κB)-dependent transcription, even in the presence of cytokine signaling. Moreover, for several tested host proteins, genetic knock-down substantially reduces viral replication. Additionally, we show for USP25 that this effect is phenocopied by the small-molecule inhibitor AZ1. Our results connect viral proteins to human genetic architecture for COVID-19 severity and offer potential therapeutic targets.